Summary of Work: Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vitro. Bacteriophage RB69 DNA polymerase supplied from a plasmid can replace the normal bacteriophage T4 DNA polymerase when the latter is mutationally inactivated, retaining high fidelity during T4 DNA replication. (The structure of the RB69 DNA polymerase has been described.) Using mutations that alter critical polymerase and exonuclease amino acids, we are characterizing the resulting changes in mutation rates using both reversion and forward-mutation tests in vivo and in vitro. Inactivating the proofreading exonuclease strongly increases mutation rates. Altering a key tyrosine residue in the polymerase active site also produces a strong mutator that preferentially generates transition mutations. Mutation rates caused by these mutator mutations are somewhat higher when measured in vivo than in vitro. The combined mutator activity of the polymerase mutator and the exonuclease-defective mutator is not much greater than either alone. Clear differences are also seen between mutator specificities in vitro and in vivo.